JP5108752B2 - Compounds and their use for the treatment of autoimmune diseases - Google Patents

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application is claims the benefit brought by U.S. Provisional Patent Application No. 60 / 681,141 of May 2005 16 with application.

  The present invention has the following formula:

[Where,
R ₁ and R ₃ are NH ₂ , F, Cl, C ₁ -C ₄ alkoxy, or a phenoxy group, but R ₁ and R ₃ are not necessarily equal;
R ₂ is H, F, Cl, NH ₂ , or NH—R—XH, where R is — (CH ₂ ) _p —, where p is 2-4.

(Wherein, q is 0 to 3) is, X is located in CH _2, NH, O or S,;
n is 0-2;
m is 0-2;
m and n are not necessarily equal to each other].

  These compounds are useful in that they can be used for the treatment of autoimmune diseases.

  Autoimmune diseases are a group of diseases in which tissue damage is associated with humoral and / or cell-mediated immune responses to body components or, in a broader sense, with immune responses against self. Or it represents one of the obstacles. The pathological immune response may be systemic or organ specific. That is, for example, self-directed immune responses can affect the joints, skin, myelin sheath that protects neurons, kidneys, liver, pancreas, thyroid, adrenal glands, and ovaries. In fact, the list of autoimmune diseases consists of over 80 disorders. Some autoimmune diseases, such as vitiligo, where skin spots lose pigmentation, are simply annoying (distressed). Most other disorders are debilitating, often progressing over time, and ultimately life-threatening. For example, systemic lupus erythematosus (SLE) is a chronic disease in which 10-15% of patients die within 10 years of diagnosis. With the exception of some autoimmune diseases, the sex ratio is biased towards women. For example, in SLE, the female to male ratio is 9: 1. In the specific case of Hashimoto's disease, where the immune system attacks the thyroid gland, the female to male ratio is 50: 1.

  It has long been known that immune complex formation plays a role in the pathogenesis and progression of autoimmune diseases. For example, in “The Pharmacological Basis of Therapeutics, 16th Edition (1980), Macmillan Publishing, p. 683” by Goodman and Gilman, inflammation in patients with arthritis consists of antigens, antibodies, and complement. It is explained that phagocytosis by leukocytes of the complex (immune complex) is probably involved. Currently, however, inflammation caused by immune complexes in the joints (arthritis), kidneys (glomerulonephritis), and blood vessels (vasculitis) is recognized as the main cause of the morbidity in autoimmune diseases ( (See Hogarth, PM et al., Annual Reports in Medicinal Chemistry 37: 17-224 (2002)). There is a correlation between increased immune complex formation and the presence of antibodies directed to the self (so-called autoantibodies), and the presence of autoantibodies is further unbound as part of the immune complex or to the antigen. In some cases (free antibodies), it can contribute to tissue inflammation. In some autoimmune diseases, the presence of free autoantibodies greatly contributes to the disease pathology. This has been clearly demonstrated in, for example, SLE (anti-DNA antibody), ITP (antibody response directed against platelets), and to a lesser extent rheumatoid arthritis (IgG-responsive rheumatoid factor). The important role of immune complexes and free autoantibodies due to the fact that the removal of immune complexes and free antibodies using specific immunosorbent methods successfully achieves the treatment of certain autoimmune diseases Has been further demonstrated. For example, the use of plasma exchange (apheresis), in which immune complexes and antibodies are removed by passing the patient's blood through an immunoaffinity [PROSORBA®] column, is related to immune thrombocytopenia in 1987. Approved by the US Food and Drug Administration in 1999 and for rheumatoid arthritis in 1999. However, currently there are no approved methods for treating autoimmune diseases that facilitate the removal of immune complexes and autoantibodies by administering drugs.

  Another aspect related to the pathogenesis and progression of autoimmune disease is the role of pro-inflammatory cytokines. Under normal circumstances, pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-1 (IL-1) play a protective role in the response to infection and cellular stress. However, pathological consequences resulting from chronic and / or excessive production of TNFα and IL-1 are the progression of many autoimmune diseases such as rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, and psoriasis It is thought that it is the basis of Other pro-inflammatory cytokines include interleukin-6, interleukin-8, interleukin-17, and granulocyte macrophage colony stimulating factor. However, TNFα is thought to be located at the tip of the proinflammatory cytokine cascade. That is, in terms of blocking certain proinflammatory cytokines, blocking TNFα provides the greatest therapeutic effect. The ability of TNFα to down-regulate other pro-inflammatory cytokines is reviewed by “Feldman, M., in Perspectives 2: 364-371 (2002)”. In fact, the effects of TNFα antagonism as a treatment option for arthritis, psoriatic arthritis, psoriasis, and Crohn's disease are REMICADE® (chimeric anti-TNFα monoclonal antibody), ENBREL® (soluble TNFαp75 Receptor fusion protein), and HUMIRA® (human anti-TNFα monoclonal antibody) have been demonstrated to be approved by the US Food and Drug Administration.

  As can be inferred from the above discussion on the etiology and progression of autoimmune diseases, their pathogenesis is complex and multifactorial. Therefore, there are many treatments available. However, most autoimmune diseases are not sufficiently suppressed by conventional treatments. Prior art therapies are not uniform in effectiveness and are often associated with moderate to substantial toxicity. Nevertheless, from the above discussion, the body can help remove immune complexes, or at least can prevent the deposition of circulating immune complexes, and / or It can be seen that there is a need for simple, clear organic compounds that inhibit the activity of TNFα while the patient is well tolerated. In summary, there is a need for effective and well tolerated treatments for chronic autoimmune diseases.

The present invention provides compounds useful for the treatment of chronic autoimmune diseases. Most autoimmune diseases are chronic symptoms that are not life-threatening at first, but gradually progress to a debilitating state. Although many treatments are available, conventional treatments are not always effective. More problematic is the accompanying toxicity, which often hinders the long-term use required in the case of chronic diseases. Current therapeutics for autoimmune diseases can be broadly divided into two groups: drugs that weaken or suppress the immune response to self; and drugs that address symptoms resulting from chronic inflammation. More specifically, conventional treatments for autoimmune diseases (e.g., primarily arthritis) are as follows:
1.Nonsteroidal anti-inflammatory drugs (NSAID):
NSAIDs include aspirin, ibuprofen, naproxen, etodolac, and ketoprofen. NSAIDs are less potent drugs and are therefore most commonly used as anti-inflammatory agents in the early stages of the disease (eg to relieve pain and swelling associated with arthritis). However, NSAIDs are associated with gastrointestinal irritation and liver toxicity. Selectively inhibit cyclooxygenase-2 (VIOXX®, CELEBREX®) or preferentially inhibit cyclooxygenase-2 to address gastrointestinal ulceration associated with the use of many NSAIDs [MOBICOX®], more selective NSAID drugs (ie, COX-2 inhibitors) have recently been developed. However, COX-2 inhibitors exhibit troublesome side effects, including gastrointestinal irritation, especially when used for longer periods.

2. Corticosteroids Corticosteroids include prednisone and dexamethasone. Corticosteroids are the most widely used anti-inflammatory agents for the treatment of rheumatoid arthritis. However, corticosteroids greatly increase the risk of infection resulting from osteoporosis, gastrointestinal toxicity, and systemic immunosuppression. Corticosteroids therefore tend to be used not for the treatment of chronic diseases but for the treatment of disease expansion (eg SLE).

3.Preventive maintenance antirheumatic drug (DMARD)
DMARDs include cytotoxic agents such as methotrexate, azathioprine, and cyclophosphamide; powerful immunosuppressants such as cyclosporin A (SANDIMMUNE (registered trademark), NEORAL (registered trademark)) and FK506 (tacrolimus); And various other drugs such as chloroquine and organic gold salts (eg gold thioglucose); DMARD is a powerful drug and can therefore be highly effective in reducing inflammation and slowing the rate of disease progression. Therefore, doctors have traditionally used DMARD as a second-line treatment after NSAIDs. However, since DMARDs are powerful drugs, they have a fairly high toxicity associated with their use. For example, cytotoxic agents prevent DNA replication that manifests with many toxic effects. Toxic effects include myelosuppression and the risk of subsequent infection and new tissue formation. The use of cyclosporine A and FK506 is limited by serious side effects, including nephrotoxicity and liver toxicity. Toxic effects associated with the use of hydrochloroquine include blindness, neuromyopathy, and gastrointestinal disorders. The most common side effect resulting from treatments using gold salts is dermatitis. However, gold poisoning can cause nephritis and bone marrow suppression.

4. The biologics biologics, a recombinant protein REMICADE (R), ENBREL (R), and HUMIRA (R) (both of which are the TNFα targeting); interleukin - KINERET (R) targeting 1; Amevive (CD2 surface glycoprotein) targeting T cells; and RAPTIVA (R) (anti-CD11a antibody) targeting T cells; However, recombinant proteins and particularly recombinant antibodies are difficult to create for widespread use and have toxic side effects associated with use. Toxicity involves a potential immunological response, especially in the case of long-term use required for chronic symptoms. In addition to the well-known HAMA (human anti-mouse antibody) response associated with chimeric and humanized antibodies, antibody-mediated (ADCC-mediated and complement-mediated) cytotoxic mechanisms can cause side effects. Most recently, it has been found that antibodies can convert oxygen molecules into hydrogen peroxide and ozone, regardless of the source or antigen specificity ["Wentworth, P. et al., Science." 293: 1806-1811 (2001) and 298: 2195-2199 (2002)]. This can cause cell and tissue damage and can exacerbate the treatment of autoimmune diseases with long-term use. For example, it has been shown that the production of hydrogen peroxide and ozone by antibodies can be associated with an inflammatory response (the so-called Arthus reaction) in rats. The strong anti-TNFα activity of the REMICADE® antibody increases the risk of opportunistic infections, including tuberculosis, histoplasmosis, listeriosis, and interstitial plasma cell pneumonia.

  The object of the present invention is to provide novel compounds for use in treating autoimmune diseases. Autoimmune diseases (especially chronic diseases such as arthritis and SLE) can be treated by administering the compounds described herein to a mammal (preferably a human). Thus, according to the present invention, clearance of immune complexes can be promoted, or deposition in body organs (eg, kidneys) can be restricted, and / or the pro-inflammatory effect of TNFα can be inhibited. Disubstituted or trisubstituted purines, and pharmaceutical compositions thereof, are provided.

  In one embodiment of the invention, these purine compounds affect both aspects of the inflammatory process (immunocomplex and TNFα). The therapeutic benefits arising from the dual mechanism of action appear with respect to an improved toxicity profile. That is, the purine compounds described in the present invention are not potent inhibitors of TNFα and do not completely eliminate immune complexes. While TNFα plays a role in protecting against infections, immune complexes play a role in feedback mechanisms that modulate the immune response (idiopathic determinants). Therapeutic efficacy arises from the additive effects of the two mechanisms of action. Further, toxicity due to chronic disease therapeutics and / or other drugs used in combination can be at least reduced or avoided.

  In other embodiments of the invention, the purine compound affects only one embodiment of the inflammatory process. That is, these compounds affect either immune complexes or TNFα. If purine compounds affect the removal of immune complexes or prevent the deposition of immune complexes, such compounds can be used for arthritis, systemic lupus erythematosus (SLE), immune thrombocytopenia (ITP), It is believed to be particularly useful for the treatment of glomerulonephritis and vasculitis. When purine compounds inhibit TNFα, such compounds may be present in rheumatoid arthritis, psoriatic arthritis, psoriasis, Crohn's disease, inflammatory bowel disease, ankylosing spondylitis, Sjogren's syndrome, Still's disease (macrophage activation syndrome), grape It is believed to be particularly useful for the treatment of meningitis, scleroderma, myositis, Reiter syndrome, and Wegener syndrome. Of course, some purine compounds of the present invention are inflamed by biochemical mechanisms in addition to, and different from, the effects on immune complexes and / or TNFα. May affect the process. One such possible mechanism (eg, by the purine skeleton) is the effect on the adenosine receptor. However, regardless of the mechanism by which the purine compound affects the targeted autoimmune disease, the compound does not strongly affect any aspect of the inflammatory process and is therefore well tolerated for treatment. That is an important aspect of the present invention.

Further aspects of the invention will be apparent to those skilled in the art from consideration of the following description and claims, and generalizations thereto.
Detailed Description of the Invention and Specific Embodiments
The present invention has the following general formula:

[Where,
R ₁ and R ₃ are NH ₂ , F, Cl, C ₁ -C ₄ alkoxy, or a phenoxy group, but R ₁ and R ₃ are not necessarily equal;
R ₂ is H, F, Cl, NH ₂ , or NH—R—XH, where R is — (CH ₂ ) _p —, where p is 2-4.

(Wherein, q is 0 to 3) is, X is located in CH _2, NH, O or S,;
n is 0-2;
m is 0-2;
m and n are not necessarily equal to each other], or a pharmaceutically acceptable derivative of the compound.

n and m are not equal (e.g., n = 0, m = 2), R ₁ is NH ₂ and R ₃ is NH ₂ , F, or Cl, or any combination thereof. Is a preferred embodiment of the present invention. More preferably, R ₁ is meta-NH ₂ , F, or Cl.

  Particularly preferred are the following compounds:

  The compounds of the present invention promote the clearance of immune complexes by phagocytosis, or deposit immune complexes in body organs and biological tissues by the ability to weaken the binding of immune complexes to the surface of organs and tissues. Limit. The mechanism by which immune complexes bind to various surfaces may involve binding to cell surface Fc receptors. Fc receptors are inflammatory leukocyte glycoproteins that bind to the Fc (tail) portion of immunoglobulins. Fc receptors are also present on many tissues and serve as sites for binding of immune complexes to the tissue surface and subsequent deposition. For example, deposition of complex-containing autoantibodies on kidney tissue by binding to Fc receptors is believed to induce an inflammatory response typical of SLE (which can cause glomerulonephritis). Well-characterized Fc receptors include FcγRI, FcγRII, and FcγRIII (IgG receptor); FcεRI (IgE receptor); and FcαRI (IgA receptor); Interestingly, Staphylococcal aureus protein A is a cell surface bacterial protein that can bind to the Fc (tail) portion of most antibodies. For example, protein A binds to human IgGl, IgG2 and IgG4 immunoglobulins. More importantly, it has been known for many years that protein A can inhibit the binding of IgG antibody-containing immune complexes to Fc receptors. For example, “Sulica, A., et al. Immunology 38: 173-179 (1979)” states that protein A inhibits the binding of IgG-containing immune complexes to Fc receptors, but IgG to lymphocytes and macrophages. Reported increased binding.

  More recently, the availability of mice lacking Fc receptors (γ chains) has been used to mediate the effects of IgG Fc receptors (FcγRs) in mediating effector responses seen in autoimmune diseases such as SLE and rheumatoid arthritis. It has become possible to verify the original role (see “Marino, M., et al. Nature Biotechnology 18: 735-739 (2000)). In more detail, these reporters have reported that FcγR It states that drugs that can prevent the binding of immune complexes to the body should improve SLE, which is a special strain of mice (MRL / lpr) that develops a syndrome similar to human SLE. Treatment with a peptide that binds to the Fc portion of IgG provided experimental support for this report.The survival rate of treated animals (80%) was significantly higher than untreated animals. "Hogarth , PM, Current Opinion in Immunology 14: 798-802 (2002) FcγR acts early in the inflammatory process, by contact (Engagement) immune complexes is a strong signal on the release of pro-inflammatory cytokines (e.g. TNF [alpha]), which is stated that. If the compounds of the invention affect some aspect of immune complex clearance and deposition, the compounds of the invention will likely do so by their ability to mimic protein A. That is, such a compound can bind to the Fc portion of human IgG. This has been confirmed by the ability to inhibit protein A binding to human IgG and has been confirmed by an in vitro competitive ELISA. By binding human IgG to the Fc moiety in a manner similar to protein A, such protein A mimetic compounds may inhibit the binding of IgG-containing immune complexes to FcγR. This therefore prevents the deposition of immune complexes, which not only promotes their clearance, but also reduces the release of proinflammatory cytokines.

  In addition or alternatively, the compounds of the present invention may inhibit the pro-inflammatory action of TNFα. Unlike the currently approved recombinant anti-TNFα monoclonal antibodies [REMICADE®, HUMIRA®] and soluble TNFα receptors [ENBREL®], the compounds of the present invention have p55 TNFα receptor (CD120a ) And p75 TNFα receptor (CD120b). Nevertheless, the compounds of the present invention inhibit the action of TNFα, as confirmed by the ability to inhibit TNFα-induced apoptosis / cytotoxicity in the WEHI 164 (13var) mouse cell line. There is. The compounds of the present invention may further inhibit TNFα production, as confirmed by the ability to inhibit LPS-induced TNFα production in the J774A 1 mouse cell line.

  TNFα is produced by many cell types including fibroblasts and numerous immune cell subsets. Examples of the latter include macrophages, mononuclear cells, B cells, T cells, and mast cells. TNFα is a pleiotropic molecule produced in response to various stimuli and can affect most cell types. Under normal circumstances, low levels of serum TNFα provide protection against pathogens, tumors, and tissue damage. Thus, in terms of long-term or continued use of the compounds of the invention as therapeutic agents, these compounds are not potent inhibitors of TNFα action or production, and strongly bind TNFα to its receptor. Not inhibiting is one embodiment of the present invention. The effectiveness of the compounds of the present invention for long-term use has been demonstrated by treatment of NZBW / F1 mice (another model for human SLE) using the compounds of the present invention for about a year (any serious toxicity Not observed).

  Similar to the biologics described above, other TNFα inhibitors exhibit toxicity that requires limited long-term or continued use. For example, thalidomide (N-phthalimide-glutarimide) is a synthetic anti-inflammatory agent that inhibits TNFα synthesis. However, clinical trials on patients with rheumatoid arthritis have been unsuccessful in most cases due to unacceptable toxicity. Drowsiness, peripheral neuropathy, and severe rashes were observed as serious side effects. Many drugs commonly used as immunosuppressants (eg, cyclosporin A and methotrexate) exhibit inhibitory properties against TNFα but cannot be used in long-term use due to toxicity.

  Indeed, the role played by TNFα in many autoimmune diseases is crucial (as evidenced by the therapeutic success of recently approved biologics, along with the lack of more non-toxic effective drugs that can be used for long-term treatment. Therefore, it has been studied based on a number of approaches for the inhibition of TNFα. These approaches include inhibitors of phosphodiesterase IV, agonists of adenosine, matrix metalloproteinase inhibitors (e.g. inhibitors of TACE), signal transduction inhibitors (e.g. p38 MAP kinase), and inhibitors of transcription regulators (e.g. NFκB) Includes the quest for Therefore, it is clear that there is a need for compounds that effectively inhibit the action of TNFα but that can be used for a long time for the treatment of chronic autoimmune diseases.

  The present invention provides novel compounds defined by the general formula useful for the treatment of chronic autoimmune diseases. These compounds may promote immune complex clearance by phagocytosis, or by the ability to counteract the binding of immune complexes to the surface of organs and tissues, In some cases, accumulation is suppressed. In this case, such compounds would treat autoimmune diseases (e.g. arthritis, SLE, ITP, glomerulonephritis, and vasculitis) where the immune complex plays an important role in disease pathology Is particularly useful. The compounds of the present invention may further inhibit the pro-inflammatory effect of TNFα. In this case, such compounds may be used in autoimmune diseases where inhibition of TNFα biological activity is important for disease pathology (e.g. rheumatoid arthritis, psoriatic arthritis, psoriasis, Crohn's disease, inflammatory bowel It is particularly useful for the treatment of diseases, ankylosing spondylitis, Sjogren's syndrome, Still's disease (macrophage activation syndrome), uveitis, scleroderma, myositis, Reiter's syndrome, and Wegener's syndrome. It is a preferred embodiment of the present invention that these compounds mimic the activity of bacterial protein A, thereby promoting immune complex clearance. In any case, it is not intended that the scope of the present invention be limited by the mechanism by which the inflammatory condition indicative of autoimmune disease occurs. Indeed, the improvement of autoimmune disease using the compounds of the present invention may occur by less clear or unknown mechanisms, but the improvement is confirmed by in vivo activity shown in appropriate animal models. Yes. Therefore, the mechanism by which the efficacy of the compound appears is not an important aspect of the present invention, nor is it a limiting aspect. Importantly, however, is the fact that the compounds of the present invention are less toxic and can therefore be administered accordingly for the treatment of chronic autoimmune diseases.

  The compounds of the present invention include all pharmaceutically acceptable derivatives (eg, their salts and prodrugs) and analogs, as well as all geometric isomers and enantiomers. Formulations of the active compounds of the present invention include enteral mucosal administration (including sublingual, pulmonary, and rectal administration); parenteral administration (including intramuscular, intradermal, subcutaneous, and intravenous administration) Or a topical administration (including ointments, creams, and lotions); In particular, the compounds of the present invention can be used in alcohol or polyol solvents such as Solutol HS15 (polyethylene glycol 660 hydroxystearate available from BASF, glycerol, ethanol, etc.) or other biocompatible solvents such as dimethyl Sulphoxide (DMSO) or Cremophor EL (commercially available from BASF)]. Formulations can be supplied as appropriate in individual unit dosage forms as needed, or can be made by any of the methods well known in the pharmaceutical formulation industry. Any of these methods include the step of bringing into association the active pharmaceutical ingredient with a liquid carrier or a finely divided solid carrier or both as required. Where appropriate, the formulations can be adapted to provide a sustained release of the active pharmaceutical ingredient. Sustained release formulations well known in the art include the use of bolus or sustained infusion of biocompatible polymers or liposomes.

  Proper selection of dose, timing of administration, formulation, and route of administration avoids the goal of achieving a favorable response in mammals (i.e. efficacy), and other hazards to excessive toxicity or toxicity (i.e. safety) Based on the goal. Thus, “effective” means normal manipulation of the disease (eg adrenal gland, eye, joint, kidney, liver, lung, pancreas, nervous system, Reducing or ameliorating tissue damage associated with the immune response to the skin and organs and tissues such as the thyroid; repairing immunological conditions, or pathological disorders in mammals / Normalizing conditions (such as antibody force values, immune cell subsets, signaling by cytokines and chemokines, and antibody-antigen immune complexes); removing free antibodies and / or antibody-antigen immune complexes from circulation; self Including laboratory indicia of immune disease (concentration and absolute amount of soluble mediators of inflammation, presence of autoantibodies, and cell proliferation, etc .; and combinations thereof) It represents the selection. In particular, the adverse effects of conventional treatment with anti-TNFα can be avoided.

  The amount of compound administered is, for example, the biological activity and bioavailability of the compound (e.g., half-life, stability, and metabolism in the body); the chemical properties of the compound (e.g., molecular weight, hydrophobicity, and solubility); administration Dependent on factors such as route and dosing schedule; and similar factors; In addition, the specific dose level to be achieved for a particular patient depends on a variety of factors including age, health status, medical history, weight, combination with one or more other drugs, and severity of the disease. It will be understood that it depends.

  The term “treatment” or “treating” refers to one or more autoimmune diseases in a mammal (eg, a human) that is affected or at risk of developing a disease, among others. Represents reducing or relieving symptoms. For a patient, the improvement, worsening, remission, or progression of symptoms can be examined either objectively or subjectively. “Treatment” may further include combinations with other current therapies and drugs (eg, anti-inflammatory drugs, antibodies such as TNFα, drugs that bind to soluble receptors, NSAIDs, corticosteroids, DMARDs). Thus, combination therapy can be performed. In such embodiments, by reducing the amount or concentration of the additional agent used, compared to a treatment that does not include a compound of the present invention, a substantially equivalent effect on the patient is obtained while maintaining a long-term effect. It is preferred to at least reduce or avoid toxicity due to treatment or additional agents.

  It goes without saying to those skilled in the art that the treatment methods described here can be applied not only to treating and also preventing diseased autoimmune diseases and chronic autoimmune diseases. It will be appreciated that the amount of the compound of the invention required for treatment will depend not only on the type of compound used in the treatment, but also on the route of administration, the nature of the autoimmune disease to be treated, and the patient. Varies with age and general health. The dose administered will ultimately be at the discretion of the physician. In general, however, doses will range from about 0.1 to about 200 mg / kg body weight per day. The dose is preferably in the range of about 1 to about 100 mg / kg body weight per day. More preferably, the dose ranges from 2 to 50 mg / kg body weight per day. The dosage unit per day may be 10 mg or more, 100 mg or more, 10 g or less, 40 g or less, or any range in between.

  Finally, and if necessary, the compounds of the invention can be used in combination with other therapeutic agents for autoimmune diseases well known in the art. Other prior art therapeutic agents include non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, aspirin, naproxen, etodolac, and ketoprofen); corticosteroids (e.g., hydrocortisone, pregnisone, and dexamethasone) Preventive maintenance antirheumatic drugs (DMARDs) (e.g., cytotoxic drugs such as methotrexate and azathioprine, immunosuppressants such as cyclosporine and FK506, hydrochloroquine, and organogold salts); and biologics; as shown There are the aforementioned therapeutic agents. The individual components of such a combination can be administered sequentially or simultaneously in separate or combined pharmaceutical formulations. Alternatively, new pharmaceutical formulations can be created to contain combinations of the compounds of the present invention with conventional therapeutics for autoimmune diseases.

  The compounds of the present invention can also be used as affinity agents for binding antibodies (eg, human isotypes such as IgM, IgD, IgA1, IgA2, IgE, IgG1, IgG2, IgG3, and / or IgG4). . Free (ie, not bound to antigen) antibodies and / or antibody-antigen immune complexes can be specifically bound by such affinity agents. Large affinity complexes can be isolated by selective precipitation, differential centrifugation, or identified by aggregation assays. However, one or more compounds may be bound to an insoluble carrier material (e.g., agarose, dextran, cellulose, polyacrylamide, other polymeric materials, silica, and glass), preferably directly by a covalent bond or indirectly through a linker. It is preferable to fix to. The compounds of the present invention can be synthesized in situ on the support or can be synthesized via an activated organic linker. If desired, the linker may be cleavable (eg, with a reducing agent or site-specific protease) so that the compound (including and not including the bound antibody) can be pulled away from the carrier. For example, one or more compounds of the invention can include a glass slide, a multiwell plate, an optical fiber, a protein chip, or a test tube for assay or analysis; a tissue culture dish for incubating cells or antigens; and magnetic beads, It can be covalently bound to a support in the form of a porous membrane, or a chromatographic medium for separation. An antibody or other Fc-containing material is conjugated (i.e. isolated) to one or more compounds of the invention and then separated from unbound material as necessary (washing and multiple binding under different conditions) Fc-containing materials can be purified. For example, binding conditions vary depending on ionic strength (for example, salt concentration) and pH, and Fc-containing substances can be released using this.

The following examples further illustrate the practice of the present invention, but the present invention is not limited to these examples.
General synthetic sequences for preparing compounds useful in the present invention are shown schematically in Schemes 1 and 2. Scheme 1 illustrates the synthetic route used to obtain the disubstituted purine derivatives described herein, and Scheme 2 illustrates the synthetic method for obtaining the trisubstituted purine derivatives described herein. ing.

measurement:
All HPLC chromatograms and mass spectra were recorded on an HP1100LC-MS Agilent instrument using a diode array detector. The analysis was performed by one of the following four methods: analytical C18 column (75 x 4.6 mm, 5 microns), gradient from 1% to 40% in acetonitrile-water system containing 0.01% TFA. 6 min, flow rate 2 ml / min (method 1); analytical C18 column (75 x 4.6 mm, 5 microns), 6% gradient in acetonitrile-water system containing 0.01% TFA with a gradient from 10% to 99% Min, flow rate 2 ml / min (method 2); analytical C18 column (75 x 4.6 mm, 5 microns), 6 min with a gradient from 15% to 99% in acetonitrile-water system containing 0.01% TFA, Flow rate is 2 ml / min (method 3); or 6 minutes with a gradient from 1% to 20% in an acetonitrile-water system containing an analytical C18 column (75 × 4.6 mm, 5 microns), 0.01% TFA, The flow rate is 2 ml / min (Method 4).

Example 1 (representative example of Scheme 1): N- {9- [2- (4-aminophenyl) ethyl] -9H-purin-6-yl} benzene-1,3-diamine dihydrochloride (compound Synthesis of 1)

  A solution obtained by dissolving 4-aminophenethyl alcohol (3.0 g, 21.8 mmol) in tetrahydrofuran (100 ml) was added to di-tert-butyl dicarbonate (5.2 g, 23.6 mmol) and triethylamine (4.7 ml, 32.8 mmol). Was added at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with water (100 ml) and ethyl acetate (100 ml). The aqueous layer was extracted with ethyl acetate (100 ml). The organic layer was dried over sodium sulfate and filtered. The protected amine was obtained as a white solid (4.3 g, 83%). This compound (1.7 g, 7.4 mmol) and triphenylphosphine (1.9 g, 7.4 mmol) were mixed with 6-chloropurine (761 mg, 4.9 mmol) in dry tetrahydrofuran (13 ml) at room temperature. Added at. The solvent was removed by evaporation from the resulting mixture. Dry tetrahydrofuran (13 ml) was added and the suspension was cooled to 0 ° C., followed by dropwise addition of diethylazodicarbosylate (891 μl, 5.7 mmol). After 16 hours at room temperature, the solution was concentrated under reduced pressure. The crude residue was purified on a Biotage® 25M column (silica, hexane / AcOEt 95: 5-65: 35) to give N-9 alkylated purine as a white solid (1.8 g, quantitative).

To a suspension obtained by mixing 1,3-phenylenediamine (8.2 g, 75.4 mmol) in methylene chloride (21 ml), di-tert-butyl dicarbonate (2.7 g, 12.6 mmol) was added to methylene chloride (130 ml). The solution obtained by dissolving in 1) was added dropwise at room temperature over 1 hour. The solution was stirred overnight at room temperature. After reacting for 18 hours, the solvent was removed from the solution by evaporation under reduced pressure. The residual oil was dissolved in ethyl acetate (50 ml) and washed with 2N sodium carbonate (50 ml). The aqueous layer was extracted with ethyl acetate (2 × 50 ml). The combined organic layers were washed with brine (50 ml), dried over sodium sulfate, filtered and the solvent was removed by evaporation. The crude residue was purified on a Biotage® 40S column (silica, hexane / AcOEt 95: 5 to 1: 1) to give N-1-tert-butyloxycarbonyl-1,3-phenylene-diamine as white Obtained as a solid (2.4 g, 93%). This compound (40 mg, 0.2 mmol) and diisopropylethylamine (50 μl, 0.3 mmol) were obtained by dissolving 6-chloro-N-9-alkylated purine (35 mg, 0.1 mmol) in n-butanol (2.0 ml). To the solution at room temperature. After reacting at 90 ° C. for 48 hours, the brown solution was concentrated under reduced pressure. The crude residue was purified by Biotage® 12M column (silica, hexane / AcOEt 6: 4 to AcOEt / MeOH 8: 2) to give the di-substituted purine as a brown solid (32 mg, 63%). To a solution of this material (32 mg, 0.06 mmol) dissolved in dichloromethane (2.0 ml) was added a solution of 4N HCl in dioxane (2.0 ml) at room temperature. After reacting at 25 ° C. for 3 hours, the solution was concentrated under reduced pressure and dried under reduced pressure for 16 hours to obtain Compound 1 as a light brown solid. Product yield: 23 mg (94%); R _f = 0.4 (AcOEt / MeOH 95: 5);

Example 2 (representative example of Scheme 2): N6- (3-amino-phenyl) -9- [2- (4-amino-phenyl) -ethyl] -N2-cyclopropylmethyl-9H-purine-2 Of 1,6-diamine (compound 3)

To a solution obtained by dissolving 2-amino-6-chloropurine (5.0 g, 29.5 mmol) in a solution obtained by dissolving tetrafluoroboric acid in water (100 ml), sodium nitrite (3.5 g, A solution obtained by dissolving 50 mmol) in water (160 ml) was added dropwise at −15 ° C. over 1.5 hours. After 20 minutes at room temperature, the pH of the solution was adjusted to 6 using 50% aqueous sodium hydroxide. The resulting solution was concentrated under reduced pressure. The crude residue was purified by Biotage® 40S column (silica, CH ₂ Cl ₂ / MeOH 9: 1) to give 2-fluoro-6-chloropurine (2.3 g, 52%). This compound (1.5 g, 8.6 mmol) was suspended in dry tetrahydrofuran (20 ml) at 25 ° C. Subsequently, 4- (N-1-tert-butyloxycarbonyl) -aminophenethyl alcohol (3.4 g, 14.5 mmol) and triphenylphosphine (3.8 g, 14.5 mmol) were added, and the solvent was removed from the mixture by evaporation. Dry tetrahydrofuran (20 ml) was added and the suspension was cooled to 0 ° C. before diethyl azodicarboxylate (1.5 ml, 9.8 mmol) was added dropwise. After reacting at room temperature for 16 hours, the solution was concentrated under reduced pressure. The crude residue was purified by Biotage® 25M column (silica, hexane / AcOEt 95: 5-75: 25) to give N-9 alkylated purine as a white solid (2.9 g, 87%) . A solution obtained by dissolving alkylated 2-fluoro-6-chloropurine (3.0 g, 7.7 mmol) in n-butanol (15 ml) was added to N-1-tert-butyloxycarbonyl-1,3-phenylenediamine. (1.8 g, 8.8 mmol) and diisopropylethylamine (2.7 ml, 15.3 mmol) were added. After reacting at 65 ° C. for 48 hours, the brown solution was concentrated under reduced pressure. The crude residue was purified by Biotage® 40M column (silica, hexane / AcOEt 65: 45-0: 1) to give the fluoropurine derivative as a brown solid (2.8 g, 63%). To a solution obtained by dissolving this product (2.8 g, 4.9 mmol) in n-butanol (15 ml), aminomethylcyclopropane (1.2 g, 17.1 mmol) and diisopropylethylamine (1.7 ml, 9.8 mmol) were added at room temperature. Added at. After reacting at 110 ° C. for 48 hours, the brown solution was concentrated under reduced pressure. The crude residue was purified on a Biotage® 40M column (silica, hexane / AcOEt 60:40 to AcOEt / MeOH 98: 2) to give the protected tri-substituted purine as a white solid (2.3 g, 73%). To a solution obtained by dissolving this compound (2.08 g, 3.38 mmol) in dichloromethane (10 m) was added 4N HCl in dioxane (10 ml) at room temperature. The reaction mixture was stirred at 25 ° C. for 5 hours, and the solution was concentrated under reduced pressure. The solid was then dried in vacuo for 16 hours to give compound 3 as a brown solid. Product yield: 1.4 g (quantitative); R _f = 0.1 (CH ₂ Cl ₂ / MeOH 98: 2);

Example 3: N6- (3-amino-phenyl) -9- [2- (4-amino-phenyl) -ethyl] -9H-purine-2,6-diamine (compound 2)
The above compound was prepared as described in Example 1 starting from 2-amino-6-chloropurine.

Example 4: 6- {6- (3-Amino-phenylamino) -9- [2- (4-amino-phenyl) -ethyl] -9H-purin-2-ylamino} -hexan-1-ol (compound Four)
The above compound was prepared as described in Example 2 except that 6-aminohexanol was used instead of aminomethylcyclopropane.

Example 5: N6- (3-amino-phenyl) -9- [2- (4-amino-phenyl) -ethyl] -N2-cyclopropyl-9H-purine-2,6-diamine (compound 5)
The above compound was prepared as described in Example 2, except that cyclopropylamine was used in place of aminomethylcyclopropane.

Example 6: N6- (3-amino-phenyl) -9- [2- (4-amino-phenyl) -ethyl] -N2-tert-butyl-9H-purine-2,6-diamine (compound 6)
The above compound was prepared as described in Example 2 except that tert-butylamine was used in place of aminomethylcyclopropane.

Example 7: N- [9- (4-Amino-benzyl) -9H-purin-6-yl] -benzene-1,3-diamine (Compound 7)
The above compound was prepared as described in Example 1 except that 4-nitrobenzyl bromide and potassium carbonate were used in place of 4- (N-1-tert-butyloxycarbonyl) -aminophenethyl alcohol. did. Reduction of the nitro group was performed using 10% Pd / C and ammonium formate.

Example 8: 2- {6- (3-Amino-phenylhumino) -9- [2- (4-amino-phenyl) -ethyl] -9H-purin-2-ylamino} -ethanol (Compound 8)
The above compound was prepared as described in Example 2, except that 2-aminoethanol was used in place of aminomethylcyclopropane.

Example 9: N- {9- [2- (4-Benzyloxy-phenyl) -ethyl] -2-fluoro-9H-purin-6-yl} -benzene-1,3-diamine (Compound 9)
The above compound was prepared as described in Example 1 starting from 2-fluoro-6-chloropurine.

Example 10: N6- (3-amino-phenyl) -9- [2- (4-amino-phenyl) -ethyl] -N2- (2,2-dimethyl-propyl) -9H-purine-2,6- Diamine (Compound 6)
The above compound was prepared as described in Example 2, except that neopentylamine was used in place of aminomethylcyclopropane.

Example 11: N6- (3-amino-phenyl) -9- [2- (4-amino-phenyl) -ethyl] -N2-isobutyl-9H-purine-2,6-diamine (compound 11)
The above compound was prepared as described in Example 2, except that isobutylamine was used in place of aminomethylcyclopropane.

Example 12: N- {9- [2- (3-Amino-phenyl) -ethyl] -9H-purin-6-yl} -benzene-1,3-diamine (Compound 12)
The above compound was prepared as described in Example 1 except that 3-nitrophenethyl alcohol was used instead of 4- (N-1-tert-butyloxycarbonyl) -aminophenethyl alcohol. Reduction of the nitro group was performed using 10% Pd / C and ammonium formate.

Example 13: N6- (3-amino-phenyl) -9- [2- (4-amino-phenyl) -ethyl] -N2-cyclopentyl-9H-purine-2,6-diamine (Compound 13)
The above compound was prepared as described in Example 2 except that cyclopentylamine was used in place of aminomethylcyclopropane.

Example 14: N- {9- [2- (4-Fluoro-phenyl) -ethyl] -9H-purin-6-yl} -benzene-1,3-diamine (Compound 14)
The above compound was prepared as described in Example 1 except that 4-fluorophenethyl alcohol was used instead of 4- (N-1-tert-butyloxycarbonyl) -phenethyl alcohol.

Example 15: ability of compounds to mimic protein A (confirmed by protein A competitive binding ELISA)
As described above, this assay evaluates the ability of the exemplified compounds to mimic protein A. Such compounds can bind to the Fc portion of human IgG (as confirmed by inhibition of protein A binding to human IgG). The Protein A competitive binding ELISA assay was performed on the MAXISORP® surface of a 96 well plate to increase protein A binding to the bottom of the plate. Wells were coated with 100 μl protein A (0.8 μg) and incubated at 4 ° C. overnight. After incubation, unbound protein A was removed by washing 3 times with phosphate buffered saline (PBS). The plate coated with 100 μl per well of a 2% solution of bovine serum albumin (BSA) was then incubated at 37 ° C. for 1 hour to block non-specific protein binding. After incubation, the plate was washed 3 times with PBS. 50 μl of compound or protein A (diluted in PBS or PBS-20% DMSO at the appropriate concentration) was added to the wells followed by 50 μl of peroxidase conjugated human IgG (HRP-IgG). After 1 hour incubation at 37 ° C., the plate was washed 3 times with PBS to remove unbound HRP-IgG. Bound HRP-IgG is detected by incubating for 20 minutes in the dark at room temperature using 100 μl of 2,2'-azino-di- [3-ethylbenzothiazolinesulfonate] diammonium salt crystal (ABTS) solution did. The plate was then read at 405 nm with a BIO-TEK® EL800 Universal Microplate Reader. Analyze the data in a Microsoft® Excel® spreadsheet and calculate the compound concentration (IC ₅₀ ) that inhibits protein A binding by 50% using Prism® software. (Shown in Table 1).

Example 16: Effect of compounds on LPS-induced TNFα production in mouse J774A-1 cell line The effect of compounds on TNFα production was determined by ELISA using J774-1 cells stimulated by lipopolysaccharide (LPS). It was measured. J774-1 cells were cultured with and without LPS and compounds. The cells were cultured at 37 ° C. for 24 hours, and then the supernatant was collected, and the concentration of TNFα was examined by ELISA recommended by the manufacturer (BD Bioscience). The data was analyzed with Microsoft® Excel® spreadsheet and the compound concentration (IC ₅₀ ) that inhibits TNFα production by 50% was calculated using Prism® software ( (Shown in Table 2).

Example 17: Effect of Compound 1 on Oxazolone-Induced Delayed Type Hypersensitivity (DTH) Compounds were tested for their ability to treat oxazolone-induced delayed type hypersensitivity (DTH) in mice. On day 0, mice were sensitized with 100 μl of 5% acetone solution of oxazolone. On days 0, 1 and 2, mice were treated with vehicle (control standard), methotrexate (MTX; positive control standard), or intravenous administration (at 50 mg / kg body weight) of a compound of the invention. . The mice were attacked by applying 50 μl of oxazolone on the surface of the right ear of the mouse (first attack, day 3; second attack, day 10). Ear thickness was measured on days 4-7 and 11-14. Redness and crust formation were observed. On day 14, the mice were sacrificed. T _DTH (CD4) cells play an important role in regulating the strength of the DTH response.

  As shown in Table 3, Compound 1 causes a significant reduction in inflammation (as seen by the smaller ear thickness). Compound 1 alone has potency equivalent to methotrexate. Compound 1 further reduces redness, crust formation, and ear swelling.

  The effect of oral administration of Compound 1 was examined according to the protocol described above except that Compound 1 was orally administered from 0 to 13 days at 50 mg or 150 mg per kg body weight. The positive control was hydrocortisone. FIG. 1 shows the effect of Compound 1 on DTH. Compound 1 causes a significant reduction in inflammation (as seen by the smaller ear thickness in both attacks 1 and 2).

Example 18: Effect of Compound 1 on Freund's Adjuvant-Induced Arthritis (AIA) Injection of a lyophilized Mycobacterium butyricum suspension in mineral oil into the foot pad Induced AIA in female Lewis rats. Arthritis development was monitored for 3 weeks after adjuvant injection. Inflammation was maximal on day 3 after adjuvant administration. Immune activation appears to occur about day 14. Compounds were injected intravenously 3 days, 2 days, and 1 day before adjuvant injection, and compounds were intravenously injected 10 days, 11 days, and 12 days after adjuvant injection. Body weight was recorded. The onset of disease was monitored using the arthritic index, which is one measure for inflammation (edema), redness, and joint stiffness. The degree of arthritis was examined by measuring the two vertical diameters of the ankle joint in the mediolateral plane and the dorsoventral plane using a caliper. The joint circumference in millimeters was then calculated using a geometric formula. The incidence and severity of arthritis were evaluated. Incidence is defined as the number of rats with clinical evidence for joint inflammation during the study period.

  As shown in FIG. 2, 100% of the animals quickly developed synovitis. A significant reduction (20%) in arthritis severity (inflammatory index) was observed with intravenous injection of methotrexate (positive control) over 13 days. In the case of Compound 1, a significant decrease in inflammation index (up to 25%) was observed from day 1 to day 21.

  The effect of oral administration of Compound 1 was examined according to the protocol described above, except that Compound 1 was orally administered at 50 mg / kg body weight from day -3 to day 19. The positive control was indomethacin. As shown in FIG. 3, a significant reduction (10-40%) in arthritis severity (inflammatory index) was observed with oral administration of indomethacin (positive control) for more than 1 day. In the case of Compound 1 as well, a significant decrease (10-30%) in the inflammation index was observed from day 1 to day 20.

The entire contents of the patents, patent applications, and other publications cited herein are hereby incorporated by reference.
All improvements and substitutions that fall within the scope of the claims and their legal equivalents are to be embraced within the scope of the invention. A claim using the term “comprising” can include other ingredients within the scope of the claim. The present invention further includes, in place of the term “comprising”, “consisting essentially of” (ie, if it does not substantially adversely affect the practice of the invention, the claims) The term "can include other components within the scope" and claims "consisting" (ie other than impurities or non-critical agents normally associated with the present invention) Claim) using the term (including only those ingredients described in). Any of these three terms can be used to claim the present invention.

  It should be understood that the components described herein are not to be considered as limiting the claimed invention unless explicitly stated in the claims. Thus, the claims are the basis for defining the scope of legal protection permitted instead of the limitations from this specification being read into the claims. In contrast, the prior art is explicitly excluded from the present invention to the extent of the specific embodiments that precede or detract from the claimed invention.

  In addition, if a particular relationship between the limitations of a claim is not specified in the claim, such a particular relationship is not intended (e.g., an arrangement of components in a product claim or a process in a method claim). Order is not a limitation of the claims unless explicitly stated otherwise). Any possible combination and replacement of individual components disclosed herein should be considered an embodiment of the invention; similarly, generalization of the description of the invention should also be considered as part of the invention. It is.

  From the above description, it will be apparent to those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. The embodiments described herein are to be considered merely as illustrative and not restrictive. This is because the scope of legal protection given to this invention is indicated by the appended claims rather than by this specification.

Figure 2 shows the effect of oral administration of Compound 1 as compared to hydrocortisone for delayed type hypersensitivity (DTH). Figure 2 shows the effect of intravenous administration of Compound 1 as compared to methotrexate for adjuvant-induced arthritis. Figure 2 shows the effect of oral administration of Compound 1 when compared to indomethacin for adjuvant-induced arthritis.

Claims (15)

The following formula

[Where,
n is 0;
m is 2;
R ₁ and R ₃ are equal and are NH ₂ , F, or Cl ;
R ₂ is H, F, Cl, NH ₂ , or NH—R—XH, where R is — (CH ₂ ) _p —, where p is 2-4.

Where q is 0-3,
X is CH ₂ , NH, O, or S]. R ₁ is meta -NH _2, F or Cl,, R ₂ is H, A compound according to claim 1.

A compound selected from the group consisting of: 4. A compound according to any one of claims 1 to 3 capable of non-covalent binding to an antibody. 5. A composition comprising at least one compound according to any one of claims 1-4 , wherein the compound is combined with a pharmaceutically acceptable carrier. Further seen containing a recombinant protein capable of binding to human TNF [alpha], the recombinant protein, infliximab, a etanercept or adalimumab composition of claim 5. 6. The composition of claim 5 , further comprising methotrexate, an anti-inflammatory corticosteroid, a non-steroidal anti-inflammatory agent, or a combination thereof. Binding the compound of claim 4 to at least an antibody or antibody-antigen immune complex;
And separating unbound material to isolate said antibody or antibody-antigen immune complex;
An in vitro method of using the compound of claim 4 as an affinity promoter. 9. The method according to claim 8 , wherein the one or more compounds are immobilized on an insoluble carrier. A compound according to any one of claims 1-4, or a therapeutically effective amount of a composition according to any one of claims 5-7, for the treatment of patients suffering from autoimmune diseases Pharmaceutical composition . 11. The pharmaceutical composition according to claim 10 , wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, arthritis, glomerulonephritis, immune thrombocytopenia, and vasculitis. Use of a compound according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment of an autoimmune disease. 13. Use according to claim 12, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, arthritis, glomerulonephritis, immune thrombocytopenia, and vasculitis. Use of a compound according to claim 4 for binding at least to an antibody or antibody-antigen immune complex in vitro. 15. Use according to claim 14, wherein the one or more compounds are immobilized on an insoluble carrier.

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